Pharmaceutical compositions comprising disulfiram

ABSTRACT

Disulfiram (tetraethylthiuram disulfide) is shown to inhibit angiogenesis and to be useful in the treatment of angiogenesis-dependent disorders, including neoplasms, and to prevent cell hyperproliferation and formation of clots along or around medical devices.

CROSS REFERENCE TO RELATED APPLICATION

The present application is the national stage under 35 U.S.C. 371 ofPCT/IL99/00017, filed Jan. 11, 1999.

FIELD OF THE INVENTION

The present invention relates to the use of disulfiram as anangiogenesis inhibitor for the preparation of pharmaceuticalcompositions useful for the treatment of angiogenesis-dependentdisorders and to a method of treatment of said disorders.

BACKGROUND OF THE INVENTION

Bis(diethylthiocarbamoyl) disulfide or tetraethylthiuram disulfide,hereinafter disulfiram, is an inhibitor of various enzymes and achelator of heavy metals. Disulfiram is known as an alcohol deterrentand is the active ingredient of the drug Antabuse used in aversiontherapy, an adjunctive treatment for chronic alcoholism (Haley, 1979).Disulfiram has an ampiphilic nature, is soluble in water but itsolubilizes better in hydrophobic solutions such as methanol, acetone orchloroform.

Once ingested and absorbed through the intestinal tract or injectedintraperitoneally as a colloidal suspension, disulfiram is extensivelydistributed throughout the body including the brain (Faiman, 1978). Itis quickly converted into its main metabolite, diethyldithiocarbamate(DDC) (Eneanya, 1981), whereby the disulfide is reduced to a sulfhydrylgroup. The DDC produced is then further metabolized via different wayswhich include a non-enzymatic degradation to diethylamine (DEA) (Brien,1983).

Both disulfiram and DDC are potent chelators of copper, iron and zinc.Chelation of the metal portion of an enzyme by disulfiram or DDC mightlead to the inactivation of such enzyme. Thus disulfiram was shown toinhibit various zinc-containing dehydrogenases, such as aldehydedehydrogenases as well as oxidases, dopamine-β-hydroxylases andaldolases (Eneanya, 1981). Inhibition of aldehyde dehydrogenases bydisulfiram inhibits glycolysis, the tricarboxylic acid cycle and thepentose phosphate shunt.

Disulfiram was shown to interfere with induction of cancer by variouscarcinogens, often by inhibiting their metabolic activation, as shownfor bladder cancer in rats exposed toN-butyl-N-(4-hydroxybutyl)nitrosamine (Irving, 1979) or toN-butyl-N-(3-carboxypropyl)nitrosamine (Irving, 1983), for liver tumorsin rats induced by dimethyl- or diethylnitrosamine (Schmahl, 1976), forintestinal cancer induced by azoxymethane in rats (Nigro, 1978)(probably by blocking the metabolism of azoxymethane), for neoplasia ofthe large bowl induced by 1,2-dimethylhydrazine (Wattenberg, 1978), andin neoplasia of the forestomach induced by benzo(a)pyrene in mice(Borchert, 1976). Disulfiram inhibited the metabolism of the carcinogenazomethane thus offering protection from the oxymetabolite neoplasia(Fiala, 1977). Disulfiram was also shown to inhibit tumorprogression—from papilloma to squamous cell carcinoma—in the murine skinmultistage carcinogenesis model (Rotstein, 1988), to inhibittransmammary carcinogenesis induced in mice by7,12-dimethylbenz(a)anthracene (Rao, 1989) and to reduce the incidenceof mammary tumors induced in rats by N-2-fluorenylacetamide orN-hydroxy-N-2-fluorenylacetamide through inhibition of their metabolicactivation (Malejka Giganti, 1980). Since a cytosolic aldehydedehydrogenase is induced during rat hepatocarcinogenesis (Allen, 1982),the inhibitory effect of disulfiram on various carcinogens may berelated to its inhibitory effect on aldehyde dehydrogenases.

Disulfiram was shown to protect mice against ifosfamide- andcyclophosphamide-induced urotoxicity when administered simultaneouslywith said drugs without compromising their anti-tumor activity againstSarcoma 180, EL-4 leukemia or L1210 murine leukemia (Hacker, 1982;Ishikawa, 1991; Ishikawa, 1994). On the other hand, disulfiram did notshow any protection against cisplatin nephrotoxicity in humans (Verma,1990). Disulfiram protected rats against the toxic side effects of1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea (HECNU), withoutinhibiting its anti-tumor potency (Habs, 1981). Disulfiram was alsoshown to potentiate the anti-cancer activity of some agents such as tonitrogen mustard (HN2) cytotoxicity against murine leukemia at 3mg/mouse (Valeriote, 1989).

Disulfiram, together with ascorbic acid, augmented inhibition of Meth Atumor cell proliferation in vitro by increasing the intracellular oxygenfree radicals (Mashiba, 1990). In addition, disulfiram inhibitedsuperoxide dismutase in vivo (Forman, 1980; Ohman, 1986). All of thesecould result in an increase in oxygen species toxic to the cell therebymaking the cell more sensitive to damage by a variety ofchemotherapeutic agents or radiation that produce superoxide anionsspecies (Goodman, 1977). Resistance to cyclophosphamide andoxazaphosphorines is related to aldehyde dehydrogenase activity (Magni,1996; Rekha, 1994), and inhibition of this enzyme by disulfiram thusincrease sensitivity to these chemotherapies.

U.S. Pat. No. 4,870,101 (Ku and Doherty, 1989) discloses a method forinhibiting the release of interleukin-1 in animals which comprisesadministering to said animals an amount of disulfiram effective toinhibit the release of interleukin-1, thus proposing disulfiram for thetreatment of IL-1 mediated inflammations such as psoriasis, rheumatoidarthritis, diabetes and atherosclerosis.

Disulfiram, given 0.05% in diet for 2 years, did not increase any tumortype in rats (Cheever, 1990). The toxic dose for disulfiram in normalmice is about 6-10 mg/mouse/day. The LD₅₀ of disulfiram given orally inrats is 8.6 g/Kg.

None of the above publications describes or suggests the use ofdisulfiram as an inhibitor of angiogenesis.

SUMMARY OF THE INVENTION

It has now been found in accordance with the present invention thatdisulfiram inhibits angiogenesis and is able to block neovascularizationinduced subcutaneously in nude mice.

The present invention thus relates to the use of disulfiram for thepreparation of a pharmaceutical composition useful for inhibition ofangiogenesis.

The pharmaceutical composition of the invention is suitable fortreatment of angiogenesis-dependent diseases including, but not beinglimited to, ophthalmologic disorders such as diabetic retinopathy,corneal graft neovascularization, neovascular glaucoma, trachoma andretinopathy of prematurity also known as retrolental fibroplasia,dermatologic disorders such as dermatitis and pyogenic granuloma,pediatric disorders such as hemangioma, angiofibroma, and hemophilicjoints, orthopedic disorders such as nonunion fractures, neurologiccerebrovascular disorders such as arteriovenous malformation, neoplasmssuch as leukemia and solid tumors, connective tissue disorders such asscleroderma, and treatment of hypertrophic scars.

Examples of solid tumors that can be treated with disulfiram accordingto the invention include, but are not limited to, bladder, breast,cervix, ear, esophagus, kidney, larynx, liver, lung, ovary, pancreas,prostate, skin, stomach, thyroid, urethra and uterus carcinomas.

For the preparation of the pharmaceutical compositions of the invention,disulfiram is mixed with pharmaceutically acceptable carriers andconventional excipients to produce unit dosage formulations suitable foradministration. Any suitable mode of administration is envisaged by theinvention, but oral administration is preferred.

The dosage of disulfiram to be administered daily will depend on thedisorder being treated and the age, weight and condition of the patientbeing treated, and can be determined without difficulty by skilledphysicians. Based on the examples herein performed in animals, it can bededuced that dosages between 1-50 mg/person are suitable for humans.

In another aspect, the invention relates to a method for inhibitingangiogenesis in a mammal, particularly humans, which comprisesadministering to a mammal in need thereof an amount of disulfirameffective for inhibiting angiogenesis.

In still another aspect, the invention relates to the use of disulfiramto prevent cell hyperproliferation and formation of clots along oraround medical devices such as stents, catheters, cannulas, electrodes,and the like. In one embodiment, disulfiram may be systemicallyadministered to a patient in which such a device has been inserted. Inanother embodiment, the medical device is coated with disulfiram beforeinsertion in the patient, and such disulfiram-coated medical devices arealso envisaged by the present invention.

Abbreviations: BCE: bovine capillary endothelial cells; bFGF: basicfibroblast growth factor; BSMC: bovine vascular smooth muscle cells;DMEM: Dulbecco's Modified Eagle's Medium; EGF: epidermal growth factor;FCS: fetal calf serum; GPS: glutamine/penicillin/streptomycin; HB-EGF:heparin-binding epidermal growth factor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show inhibition of neovascularization in nude mice bydisulfiram (D). Agarose beads containing the angiogenic compound bFGF(10 μg/bead) were implanted subcutaneously in nude mice and theangiogenic potential of bFGF in vivo was demonstrated 4 days afterimplantation, in skin specimens (FIG. 1B). Saline, as a negativecontrol, did not induce neovascularization around the bead (FIG. 1A).Disulfiram (D) introduced systemically at 60 μg/mouse inhibited almostcompletely neovascularization induced by bFGF inside and around the bead(FIG. 1C). Bar is one mm.

FIGS. 2A-2D show in vitro inhibition of DNA synthesis by disulfiram in adose-dependent manner in BCE, BSMC, BALB/MK and C6 rat glioma cells,respectively, as measured by incorporation of ³H-thymidine into thecells. Experiments were done in triplicates and the inhibition wascalculated as percentage of DNA synthesis of non-treated control.

FIGS. 3A-3D show inhibition of DNA synthesis in a non-reversal manner bydisulfiram at various periods of time (1, 2, 4 and 24 hours) in BCE,BSMC, BALB/MK and C6 rat glioma cells, respectively, as measured by theincorporation of ³H-thymidine into the cells. Experiments were done intriplicates and the inhibition was calculated as percentage of DNAsynthesis of non-treated control.

FIG. 4 shows disulfiram-induced apoptosis in endothelial cells. BCEcells were incubated with 0.5-5 μM disulfiram (D) for 20 hours andanalyzed by FACS for the DNA content of the cells. Disulfiram induced ina dose-dependent manner a sub-diploid apoptotic population ofendothelial cells, that was not apparent in control-treated cells.Experiments were repeated twice with triplicates.

FIGS. 5A-5B show disulfiram-induced apoptosis in BCE, BSMC and C6 gliomacells, following incubation with the indicated concentrations ofdisulfiram (D) for 6 hours, as analyzed by the TUNEL method. The nucleiof BCE cells (FIG. 5A, bottom) treated with disulfiram were labeledusing the TUNEL staining method, while the nuclei of control BCE (FIG.5A, top) and of BSMC (FIG. 5B, top) and C6 glioma cells (FIG. 5B,bottom) treated with disulfiram were not labeled using the TUNELstaining method. Experiments were repeated twice with triplicates.

FIG. 6 shows inhibition by disulfiram of Lewis lung tumor metastasis inthe lungs of mice in the footpad model. Lungs from C57/BL mice fedsystemically 3 times a week with disulfiram (30 μg) were weighed 24 daysfollowing removal of the tumor-bearing leg. The weight of the lungs fromnormal mice was subtracted from that of the metastasized lungs. Comparedwith water-fed control, metastasis on the lungs of disulfiram-treatedanimals was significantly smaller (n=6) p=0.005.

FIG. 7 shows inhibition by disulfiram of Lewis lung tumor metastasis inthe lungs of mice in the i.v. model. Lungs from C57/BL mice fedsystemically 3 times a week with disulfiram (13-40 μg) were weighed 24days following injection of D122 tumor cells i.v. The weight of thelungs from normal mice was subtracted from that of the metastasizedlungs. Compared with water-fed control, metastasis in the lungs ofdisulfiram-treated animals was significantly smaller (n=6)(p=0.023-0.037). At 13-40 μg/mouse disulfiram decreased metastasis tothe lungs 6-19 fold accordingly. Experiments were repeated twice.

DETAILED DESCRIPTION OF THE INVENTION

Vascular smooth muscle cells and endothelial cells are the two celltypes constituting the blood vessel walls. Angiogenesis, the growth ofnew capillary blood vessels by sprouting from established vessels,requires the growth of vascular endothelial cells and vascular smoothmuscle cells. According to the data of the present invention, disulfiramis clearly identified as an effective inhibitor of angiogenesis.

Thus, as shown herein, disulfiram inhibited in vivo the induction of newblood vessels in the mouse skin and was effective when administeredorally. The ability of disulfiram to inhibit at low concentrations thegrowth of cultured capillary endothelial cells (BCE) suggests that thedrug acts directly on capillary endothelial cells. Moreover, theinhibition of endothelial cell growth was shown to be non-reversible.The growth of vascular smooth muscle cells (BSMC), another cellconstituent of the blood vessel wall, was also inhibited by disulfiramat low concentrations (0.5 μM). The fact that the drug induces apoptosisin capillary endothelial cells and fails to induce apoptosis in othercell types such as vascular smooth muscle cells, keratinocytes (MK),fibroblasts and C6 rat glioma cells, indicates that it has somespecificity for capillary endothelial cells. Indeed, when disulfiram wasadministered systemically at low doses of 25-60 μg/mouse, the formationof new blood vessels was specifically disrupted, while no evidence fordamage in other tissues was observed. The low concentration ofdisulfiram administered systemically when calculated for the volume of amouse (3 μM), was in the range of that used in vitro for endothelialcells (0.1-0.2 μM), especially when the metabolic processing of the drugin the body is taken into account.

As might be expected from its ability to inhibit capillary endothelialcells and BSMC at concentrations achievable in vivo, systemic treatmentof mice with disulfiram inhibited neovascularization in the skin. Thegrowth of C6 rat glioma cells in vitro was inhibited by disulfiram.Taken together with the fact that active angiogenesis is essential forthe progressive growth of solid tumors (Folkman, 1990) and that C6glioma tumor development is angiogenesis-dependent (Abramovitch, 1995;Ikeda, 1995; Niida, 1995; Plate, 1992), one could expect that C6 gliomatumor growth would be affected by disulfiram. Indeed, disulfiramsignificantly reduced both Lewis lung metastasis in the lungs and C6tumor development in vivo when administered systemically per os at lowconcentrations similar to those observed to be effective in vitro, bothfor endothelial and C6 glioma cells, suggesting that the inhibitoryactivity for C6 tumor growth and for metastasis in the lungs in vivo isinduced through inhibition of angiogenesis and of C6 glioma cell growth.Once ingested and absorbed through the intestinal tract or injectedintraperitoneally, disulfiram is extensively distributed throughout thebody including the brain (Faiman, 1978), but the mechanism through whichdisulfiram induces its inhibitory effects in vitro or in vivo is notknown. Also the reason for capillary endothelial cells being more liableto disulfiram than other cell types for induction of apoptosis, is notknown.

The results shown here demonstrate that disulfiram inhibits capillaryendothelial and vascular smooth muscle cell growth and induces apoptosisin capillary endothelial cells and that, when used systemically in mice,disulfiram inhibits angiogenesis and decreases C6 glioma tumor growth,clearly defining disulfiram as a new inhibitor of angiogenesis andshowing its potential use for therapy in angiogenesis-dependent diseasessuch as pathologies in which neovascularization is involved, includingneoplasia.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES

Materials and Methods

(a) Materials:

Disulfiram (Sigma) and mouse EGF (Collaborative Biomedical Products,Bedford, Mass., U.S.A.) were purchased. Recombinant b-FGF andrecombinant HB-EGF were kindly provided by Prof. Gera Neufeld, and byDr. Judith A. Abraham (Scios Nova Inc., Mountain View, Calif.),respectively.

(b) Cell lines:

C6 rat glioma cells were routinely cultured in DMEM supplemented with 5%FCS (Biological Industries, Israel), GPS (100 U/ml penicillin, 100 mg/mlstreptomycin (Biological Industries, Israel) and 2 mM glutamine (BiolabLtd. Israel)) and 125 μg/ml fungizone (Biolab Ltd, Israel).

Brain bovine capillary endothelial cells (BCE) and bovine vascularsmooth muscle cells (BSMC), kindly provided by Prof. Israel Vlodavsky(Hadassah Medical School, Jerusalem, Israel), were cultured at 37° C. inlow glucose DMEM (1 g/liter) supplemented with 10% calf serum (HyClone,Logan, Utah, U.S.A.), a serum-free supplement: biogro-1 (Beth Haemek,Israel) and GPS.

Bovine aortic vascular smooth muscle cells (BSMC) were cultured in lowglucose DMEM (1 g/liter) supplemented with 10% FCS (HyClone, Logan,Utah) and GPS.

The BALB/MK epidermal keratinocyte cell line, kindly provided by Dr. SAaronson (National Cancer Institute, Bethesda, Md., USA), was cultured(37° C., 10% CO₂ humidified atmosphere) in calcium-free MEM (BethHaemek, Israel) supplemented with 10% dialyzed FCS and murine EGF (5ng/ml).

(c) Measurement of DNA synthesis

C6 rat glioma cells were plated in 96-well plates (Nunc, Denmark) (5000cells per well) in DMEM with 5% FCS. After 6 hours the cells were rinsedand incubated for 48 hours in serum free medium. 5% FCS or growthfactors were then added to the cells for 24 hours (triplicates).³H-methyl-thymidine (5μCi/ml) (Rotem Ind. Ltd., Israel) was added to thecells for the last 14 hours. The cells were rinsed with 100 μl methanolfor 10 minutes, followed by 200 μl 5% trichloroacetic acid, and thenrinsed and lysed with 150 μl 0.3M NaOH. Radioactive thymidineincorporated into the DNA was determined for 1 min with 3 mlscintillation liquid (ULTIMA GOLD Packard) in a β-counter. DNA synthesisassays were performed in triplicates.

Bovine capillary endothelial cells (BCE) and bovine aortic BSMC wereplated in 24-well plates (6000 cells per well) in 500 μl DMEM mediumsupplemented with 10% Colorado calf serum (CCS) (GIBCO, USA) and GPS.After 24 hours, medium was changed to starvation medium (2% CCS, 0.5%BSA, GPS) for 24 hours. ³H-methyl-thymidine (5 μCi/ml) was added for thelast 6 hours. DNA synthesis assays were performed as described above, intriplicates. DNA synthesis assays in BALB/MK keratinocytes wereperformed as previously described (Marikovsky, 1995).-DNA synthesisassays were performed in triplicates.

Disulfiram was prepared in 0.1 mM stock solutions in DMSO. Controlsamples were incubated with the appropriate concentration of DMSO.Inhibition was calculated as percentage of DNA synthesis of control.

(d) Subcutaneous angiogenesis in nude mice

Spherical agarose beads of approximately 1 mm in diameter were formedfrom 4% low gelling temperature agarose (Sigma) in PBS containing b-FGFor HB-EGF as angiogenic agent. The candidate angiogenic agent (10μg/bead) was warmed in sterile microtest tubes to 40° C. in a dry-bathfor a few seconds. 10 μl of agarose solution (6% in saline, 45° C.) werethen added to 5 μl of the angiogenic compound and beads were formedabove ice using a 20 μl pipette tip. Beads were implanted subcutaneously1 cm away from the incision-site as reported previously formulticellular spheroids (Abramovitch, 1995) in mice anesthetized with asingle dose of 75 mg/kg ketamine+3 mg/kg xylazine (i.p.). Experimentswere carried out for 4 days in CD1 nude male mice. Each day one ml ofaqueous solutions with or without 0.1-0.25 mM (25-60 μg) disulfiram wasintroduced per os to the mice using a feeding needle. Treatment was forthree days starting from the day of bead implantation until one daybefore termination. Experiments were done in quadruplicates and repeatedthree times.

(e) Growth of C6 glioma tumors

C6 rat glioma cells (10⁶) were injected subcutaneously into the back ofthe neck of CD1 nude male mice. After 3 days, 1 ml of aqueous solutionswith or without 0.1-0.5 mM disulfiram (25-120 μg) was introduced per osto the mice using a feeding needle. Mice were orally fed three times perweek. Tumors were removed 30 days following C6 cells injection, weighed,fixed in buffered formalin and histological sections were prepared. Eachexperimental group included 8 animals, and experiments were repeatedtwice.

(f) Growth of Lewis lung carcinoma tumors

The Lewis lung carcinoma (3LL), which originated spontaneously in aC57/BL/6J (H-2^(b)) mouse, is a malignant tumor that producesspontaneous lung metastases. The metastatic clone D122, kindly providedby Prof. Lea Eisenbach (Weizmann Institute of Science, Rehovot, Israel),was used herein for tissue culture and for in vivo experiments. The cellcultures were maintained in DMEM supplemented with 10% heat-inactivatedFCS, glutamine, antibiotics, sodium pyruvate and nonessential aminoacids.

Two metastasis models were used: 1. The footpad model. 2. The i.v.model. The assay of tumor development in the footpad model andevaluation of lung metastases was done as previously described(Eisenbach, 1983). Briefly, eight mice in each experimental group wereinoculated with 2×10⁵ D122 cells in 0.05 ml PBS in the right hindfootpad. Three days following tumor cells injection, mice were treatedper os by disulfiram or saline 3 times per week. The tumors becamepalpable within 11-19 days. Local tumor growth was determined bymeasuring the footpad diameter with a calipers. At 26-40 days followinginoculation, when local tumor reached a diameter of 6-7 mm, mice wereanesthetized with a single dose of 75 mg/kg ketamine+3 mg/kg xylazine(i.p.) and the tumor-bearing leg was removed by amputation above theknee joint. To measure progress of metatases the mice were killed 24days following amputation by injecting 20 mg/mouse xylazine i.p. andlungs were weighed.

To distinguish between the effect on the migration of the cells from themain tumor and the development of a tumor from a metastatic foci, thei.v. model was used. D122 cells (5×10⁵) were injected i.v. to the tailof C57/B1 male mice and after 24 days mice were killed and their lungsweighed. Treatment with disulfiram was started 3 days following theinjection of D122 cells. Each experimental group included 8 animals, andexperiments were repeated twice.

(g) Analysis of apoptotic cells by FACS

Cells were cultured in plastic tissue culture dishes for 48 hours inpresence of growth media as described above, until reaching 40-50%confluency. Disulfiram was then added to the cells for 20 hours. Thecells were removed from the plates by EDTA-trypsin and fixed in ice-cold70% ethanol (BioLab, Israel) in −20° C. for 2 hr to overnight. The fixedcells (2-5×10⁶) were washed once with HEPES-buffered saline (HBSS) andincubated with 0.5 mg/ml RNAseH (Boehringer Mannheim). Afterwards thecells were resuspended in HBSS containing 50 μg/ml propidium iodide(Sigma) and subsequently analyzed on a FACSort flow cytometer (BecktonDickinson Inc.) using Lysis II software. For the analysis, 10,000 cellswere examined from each sample. The percentage of the hypodiploid cellswas measured (Darzynkiewicz, 1992; Afanasyev, 1993). The cell cyclehistogram was divided into four regions according to the cell cyclephases: Ap, apoptotic cells; G₁, diploid cells; S, intermediate cells;and G₂/M, tetraploid cells.

(h) TUNEL assay for apoptosis

Cells were cultured on microscope slides for 48 hours in presence ofgrowth media as described above, until reaching 40-50% confluency.Disulfiram was then added to the cells for 6 hours or for 20 hours inthe case of BALB/MK keratinocytes, fixed with 4% paraformaldehyde andwashed three times with PBS. Apoptosis was analyzed by the in situ TUNELstaining carried out as described (Wride, 1994). Briefly, microscopeslides were incubated for 15 min in 2×SSC buffer at 60° C., washed inDDW and incubated with 20 μg/ml proteinase K (Boehringer Mannheim) for15 min at room temperature. After a wash with DDW, endogenousperoxidases were inactivated by incubating the slides with 2% H₂O₂ inPBST (PBS with 0.05% Tween 20) for 10 min at room temperature. Slideswere then incubated in TdT buffer (Boehringer Mannheim) for 5 min atroom temperature, and a reaction mixture containing 5×TdT buffer and 1μl biotin-21-dUTP (Clontech, 1 mM stock) and 8 units of the TdT enzyme(Boehringer Mannheim) in total volume of 50 μl was subsequently added.The reactions were carried out at 37° C. for 1.5 hr in a humid chamber.The slides were washed in 2×SSC, DDW and finally with PBS and coveredwith 10% skim milk in PBST for 15 min. After removal of the skim milk,the sections were incubated with ABC solution from ABC kit (VectorLaboratories, Inc.) for 30 min at room temperature, washed with PBS andstained using AEC procedure (Sigma) The slides were then washed ×3 inDDW and stained with haematoxylin for 30 sec and mounted by Kaiser'sglycerol gelatin (Merck).

Example 1 In Vivo Inhibition of Neovascularization by Disulfiram

Agarose beads containing the angiogenic factor bFGF (10 μg/bead) wereimplanted subcutaneously into CD1 nude mice as described in section (d)of “Materials and Methods” above. The results are shown in FIG. 1. After4 days new blood vessels clearly developed around and inside the beadscontaining bFGF (FIG. 1B) while the control beads containing only salineappeared clear and without any new blood vessels being formed around orwithin the beads (FIG. 1A). However, when the mice were daily fed per osduring 3 days with 1 ml aqueous solution (0.1-0.25 mM) of disulfiram (D)(25-60 μg/mouse/day), angiogenesis around the beads containing bFGF wasclearly inhibited (FIG. 1C).

Example 2 Disulfiram Inhibits Capillary Endothelial Cell Proliferation

To determine whether disulfiram acts directly on endothelial cellsrather than on accessory cells such as macrophages and mast cells thatcan be responsible for the development of an angiogenic response invivo, the effect of the drug on the growth of BCE cells was examined invitro as described in section (c) of “Materials and Methods” above. DNAsynthesis in BCE cells was measured in presence of increasingconcentrations of disulfiram incubated for 24 hours with the cells. Theresults are shown in FIG. 2 At. concentrations ranging from 0.1-0.5 μM,disulfiram was able to inhibit DNA synthesis in BCE cells in a dosedependent manner, complete inhibition being achieved at 0.5 μMdisulfiram (FIG. 2A). BCE cells were shown to be more sensitive to theinhibitory activity of disulfiram than other cell types such as BALB/MKkeratinocytes (FIG. 2C), C6 rat glioma cells (FIG. 2D) or bovine aorticvascular smooth muscle cells (BSMC) (FIG. 2B). Maximal inhibitoryactivity (80%) for BSMC was at 0.5-1 μM disulfiram, while maximalinhibitory activity for C6 glioma cells or BALB/MK keratinocytes was atconcentrations 10 fold higher (2-3 μM) than those for endothelial cells(0.2-0.5 μM). At higher concentrations (>10 μM), disulfiram became lessinhibitory for all cell types examined (not shown).

To examine the time course of the effect of disulfiram and whether theeffect was reversible, cells were incubated with disulfiram for variousperiods of time (1, 2, 4 and 24 hours), then washed away and DNAsynthesis was determined. One to 4 hours of exposure to disulfiram wereenough to induce maximal inhibitory effect at concentrations rangingfrom 0.5 μM for BCE cells (FIG. 3A) to 5 μM for BALB/MK keratinocytes(FIG. 3C) and C6 glioma cells (FIG. 3D). As shown, for BCE, MK and C6glioma cells the inhibitory effect seemed to be maintained even 24 hoursfollowing short exposure to the drug. BCE cells were most sensitive: 1-2hours of incubation with as low as 0.5 μM disulfiram were enough toinduce near maximal inhibition of DNA synthesis (FIG. 3A). BSMC wereless sensitive as far as time course is concerned when incubated with 1μM disulfiram. Following 4 hours of incubation with 1 μM disulfiram, DNAsynthesis inhibition in BSMC reached only 50% (FIG. 3B). The data shownhere indicate that the damage for capillary endothelial cells, followinga short incubation of 1 to 2 hours, was maximal and that, at least in atime scale of 24 hours, this damage was non-reversible.

Example 3 Inhibition of Endothelial Cell Proliferation by Disulfiram isVia Apoptosis

To determine whether the non-reversible inhibition of capillaryendothelial cells was induced by the programmed cell death pathway,apoptosis in BCE cells was examined by means of the FACS analysis and bythe TUNEL method.

Capillary endothelial cells were grown during 48 hours to 40-50%confluency and disulfiram was then added to the cells for 20 hours asdescribed in section (g) of “Materials and Methods” above. The resultsare shown in FIG. 4. FACS analysis of the DNA content of endothelialcells incubated with 0.5-5 μM disulfiram demonstrated the appearance ofa sub-diploid apoptotic population of cells. The abundance of apoptoticcells was dose-dependent. In contrast, control non-treated endothelialcells did not undergo this DNA degradation process and most of the cellswere in the G₀/G₁ phase and some in the S and G₂/M phase. Unlikecapillary endothelial cells, BALB/MK keratinocytes treated with 4 μMdisulfiram and analyzed by FACS, did not exhibit induction of apoptosis(not shown). Typically for cells undergoing apoptosis, endothelial cellstreated with disulfiram quickly became rounded. In contrast, 3T3fibroblasts, C6 glioma cells or BSMC treated with 1 μM, 2 μM or 5 μM,respectively, of disulfiram, did not change their shape to the roundedform.

The TUNEL method was used to label the nuclei of cells undergoingapoptosis as described in section (h) of “Materials and Methods” above.The results are shown in FIG. 5. As shown in FIG. 5A, bpttom, capillaryendothelial (BCE) cells incubated for 6 hours with 1 μM disulfiram wereinduced into apoptosis. In contrast, disulfiram did not induce BSMC (1μM) (FIG. 5B, top), BALB/MK keratinocytes (5 μM) (not shown) and C6 ratglioma cells (5 μM) (FIG. 5B, bottom) into apoptosis followingincubation for 6 hours, as measured by the TUNEL method. Capillaryendothelial cells are, thus, distinct in their apparent sensitivity todisulfiram-induced apoptosis.

Example 4 Disulfiram Inhibits Lewis Lung Carcinoma and C6 Glioma TumorGrowth in Vivo

Since disulfiram was shown to be an effective inhibitor ofneovascularization in vivo as well as inhibitory to C6 glioma cellgrowth in vitro, one could expect that systemic treatment with the drugmay slow tumor development, since active angiogenesis is essential forthe progressive growth of solid tumors beyond a diameter of a fewmillimeters (Folkman, 1990).

The effect of disulfiram was thus examined in the Lewis lung carcinomafootpad model in C57/BL mice as described in section (f) of “Materialand Methods” above, using concentrations that were shown to beinhibitory for angiogenesis in vivo. Twenty-four days following theremoval of the tumor-bearing leg, lungs were weighed. Metastasis in thelungs was significantly retarded by systemic treatment with disulfiram.In the control non-treated group, 50% of the mice died before or on the24th day following the removal of the tumor-bearing leg, while nodisulfiram-treated mice died. Moreover, two out of six mice in thedisulfiram-treated group had no metastases at all. As shown in FIG. 6,treatment with disulfiram at concentrations of 30 μg/mouse lowered themetastatic load in the lungs by almost 10-fold. At higher concentrations(120 μg/mouse) there was only slight decrease in the metastatic load inthe lungs (not shown).

Similarly, the effect of disulfiram was examined in a C6 rat gliomamodel in CD1-nude mice as described in section (e) of Materials andMethods above, using concentrations that were shown to be inhibitory forangiogenesis in vivo. Tumors from mice fed systemically 3 times a weekwith disulfiram were weighed 30 days following administration of C6 ratglioma cells to CD1 nude mice. The growth of the tumors wassignificantly retarded by systemic treatment with disulfiram. Comparedwith water-fed control, tumors from animals treated with disulfiram weresignificantly smaller. Experiments were repeated twice (n=8).

As shown in Table 1, at disulfiram (DSF) concentrations of 25-120μg/mouse, tumor development was retarded by 57-38%, respectively.Interestingly, the most effective concentration was the lowest one. Thisis in agreement with the data observed that disulfiram became lessinhibitory for in vitro cell growth at high concentration (not shown).Since disulfiram inhibits angiogenesis both in vivo in mice and in vitroin C6 glioma cells, it can be assumed that tumor growth inhibitionobserved for disulfiram is the result of its dual action, one on theneovascularization of the tumor and one on the C6 glioma cells.Pathological examination of various tissues (kidney, liver, stomach,lungs and spleen), including histological sections prepared from thesetissues, revealed no effect on these tissues in the treated animals.Blood vessels examined in these tissues were also not affected.

TABLE 1 Disulfiram inhibits C6 rat glioma tumor growth in nude mice (n)Tumor weight (g)^(a) % inhibition p value^(b) Control (8) 1.37 ± 0.21 0DSF (25 μg) (8) 0.59 ± 0.08 57 p = 0.003 DSF (120 μg) (8) 0.84 ± 0.18 38p < 0.05 ^(a)Tumors were weighed 30 days following administration of C6rat glioma cells into nude mice. Indicated values are mean of (n)animals ± SEM. ^(b)Significance of difference between control andtreated animals as determined by Student's test.

Example 5 Disulfiram Inhibits Lewis Lung Metastasis in the I.V. Model

To distinguish between the effect on the migration of the cells from themain tumor and the development of a tumor from a metastatic foci, thei.v. metastastis model was used as described in section (f) of materialsand Methods above. D122 cells were injected i.v. to the tail of C57/B1male mice. Three days after, disulfiram was administered to the mice peros 3 times per week, at concentrations of 13-40 μg/mouse. As shown inFIG. 7, disulfiram inhibited 83-95% of the development of metastaticfoci in the lungs. The fact that disulfiram was highly inhibitory tolung metastasis even in the i.v. model indicates that its inhibitoryeffect does not occur on the migratory phase of the cells but ratherduring foci development.

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What is claimed is:
 1. A method for inhibiting angiogenesis in anindividual which comprises administering to an individual in need therofan amount of disulfiram effective for inhibiting angiogenesis.
 2. Themethod according to claim 1, for the treatment of anangiogenesis-dependent disease selected from the group comprising anophthalmologic or a neurologic cerebrovascular disorder, a neoplasm, andhypertrophic scars.
 3. The method according to claim 2, wherein theophthalmologic disorder is diabetic retinopathy, corneal graftneovascularization, neovascular glaucoma, trachoma, or retrolentalfibroplasis.
 4. The method according to claim 2, wherein the neurologiccerebrovascular disorder is arteriovenous malformation.
 5. The methodaccording to claim 2, wherein the neoplasm is a leukemia or a solidtumor selected from bladder, brain, breast, cervix, ear, esophagus,hemangioma, kidney, larnyx, liver, lung, ovary, pancreas, prostate,skin, stomach, thyroid, urethra and uterus carcinomas.
 6. A method forpreventing cell hyperproliferation along or around a medical devicewhich comprises coating the device with disulfiram prior to itsinsertion into a patient.
 7. The method according to claim 5 whereinsaid medical device is a stent, catheter, cannula, or an electrode.
 8. Adisulfiram-coated medical device.
 9. The disulfiram-coated medicaldevice according to claim 7, wherein the medical device is a stent,catheter, cannula, or an electrode.